Transplantation of prokaryotic two-component signaling pathways into mammalian cells
Transplantation of prokaryotic two-component signaling pathways into mammalian cells
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DOI：
10.1073/pnas.1406482111
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发表时间：
2014-11-04
影响因子：
11.1
通讯作者：
Benenson, Yaakov
中科院分区：
文献类型：
--
作者：
Hansen, Jonathan;Mailand, Erik;Benenson, Yaakov
信号通路工程是合成生物电路的一条很有前途的途径。组氨酸-天冬氨酸磷酸中继被认为是在原核生物中进化的，它们形成了双组分信号的基础。酪氨酸-丝氨酸-苏氨酸磷酸化继电器，例如MAP激酶级联，在真核生物中占主导地位。最近，一个原核双组分途径在植物物种中实现了对环境三硝基甲苯的感知。我们推断，“移植”到哺乳动物宿主的双组分通路可以提供一个正交的和多样化的工具包的各种信号处理任务。在这里，我们报告说，双组分途径可以在哺乳动物细胞培养中部分重建，并用于可编程控制基因表达。为了使这种重建，组氨酸激酶（HK）和反应调节（RR）组件的编码序列进行密码子优化的人类细胞，而RR融合的反式激活结构域。通过在最小启动子前融合DNA结合位点来提供响应性启动子。我们发现，在培养的哺乳动物细胞中，HK及其同源RR的共表达是必要的，并且足以强烈诱导基因表达，即使在培养基中没有途径的化学触发物的情况下。功能丧失型和组成型突变体的表现与预期一致。我们进一步使用双组分信号通路来实现双输入逻辑AND、NOR和OR基因调控。因此，双组分系统可以在哺乳动物细胞中以不同的能力应用，并且它们的组分可以用于大规模合成基因电路。
Signaling pathway engineering is a promising route toward synthetic biological circuits. Histidine-aspartate phosphorelays are thought to have evolved in prokaryotes where they form the basis for two-component signaling. Tyrosine-serine-threonine phosphorelays, exemplified by MAP kinase cascades, are predominant in eukaryotes. Recently, a prokaryotic two-component pathway was implemented in a plant species to sense environmental trinitrotoluene. We reasoned that "transplantation" of two-component pathways into mammalian host could provide an orthogonal and diverse toolkit for a variety of signal processing tasks. Here we report that two-component pathways could be partially reconstituted in mammalian cell culture and used for programmable control of gene expression. To enable this reconstitution, coding sequences of histidine kinase (HK) and response regulator (RR) components were codon-optimized for human cells, whereas the RRs were fused with a transactivation domain. Responsive promoters were furnished by fusing DNA binding sites in front of a minimal promoter. We found that coexpression of HKs and their cognate RRs in cultured mammalian cells is necessary and sufficient to strongly induce gene expression even in the absence of pathways' chemical triggers in the medium. Both loss-of-function and constitutive mutants behaved as expected. We further used the two-component signaling pathways to implement two-input logical AND, NOR, and OR gene regulation. Thus, two-component systems can be applied in different capacities in mammalian cells and their components can be used for large-scale synthetic gene circuits.